The inessential yeast gene MUD2 encodes a protein factor that contributes to U1 small nuclear ribonucleoprotein particle (snRNP)-pre-mRNA complex (commitment complex) formation. To identify other genes that contribute to this early splicing step, we performed a synthetic lethal screen with a MUD2 deletion strain. The first characterized gene from this screen, MSL1 (MUD synthetic lethal 1), encodes the yeast homolog of the well studied mammalian snRNP protein U2B". The yeast protein (YU2B") is a component of yeast U2 snRNP, and it is related to other members of the UIA-U2B" family, the human U2B" protein, the human U1A protein, and the yeast U1A protein. It binds in vitro to its RNA target, U2 snRNA stem-loop IV, without a protein cofactor, and the target resembles more closely the U1 snRNA binding site of the human U1A protein than it does the U2 snRNA binding site of human U2B". Surprisingly, the YU2B" protein lacks a C-terminal RNA binding domain, which is conserved in all other family members. Possible functional and evolutionary relationships among these proteins are discussed.